We are studying replication of parvovirus H-1 DNA in vitro as a model for mammalian DNA replication. An activity from uninfected cells capable of converting viral single stranded DNA to double stranded replicative form has been purified and identified as DNA polymerase gamma. Additional stimulatory factors for this reaction will be purified and characterized, and the studies will be extended to the replication of double stranded replicative form. We have shown that methotrexate and FdUrd cause expansion of the pools of dUTP (and FdUTP) and lead to incorporation of dUMP (and FdUMP) into DNA. Further studies are examining the relationship of these effects to DNA fragmentation and cell death.